The steroidogenic action of ACTH can be separated into acute and chronic aspects. The acute ACTH response (sec-min) occurs primarily at the level of one mitochondria in regulating the rate- limiting step in steroidogenesis, the conversion of cholesterol to pregnenolone. The chronic action of ACTH (hours) occurs at the level of the genome and involves synthesis of various steroidogenic enzymes and co-factors. Both the acute and chronic actions of ACTH are dependent on the cytoplasmic synthesis of protein in that both responses are blocked by cycloheximide. In addition, both the acute and chronic actions of ACTH can be mimicked by cAMP. Based on mutation studies performed with an ACTH-responsive murine adrenocortical tumor cell line (Yl), as well as ACTH receptor studies involving various adrenocortical cell-types, it is now accepted that in the adrenal cortex ACTH stimulates membrane-bound adenylate cyclase activity which leads to an increase in intracellular cAMP and the activation of cAMP-dependent protein kinase followed by steroid synthesis, The role of other protein kinases in this process such as Ca2+-regulated kinases, if such a role exists, is not well understood. No adrenocortical regulatory phosphoprotein has yet been identified; and there is no evidence that the cholesterol side-chain cleavage P450 is regulated by phosphorylation-dephosphorylation. The guinea pig is used as an animal model to examine ACTH steroidogenic action for the simple reason that it is composed of an ACTH responsive outer zone and an ACTH-unresponsive inner zone. In this model, adenylate cyclase activation and cAMP formation in response to ACTH are similar for the two zones suggesting that in the inner zone a defect has developed beyond the formation of cAMP. When cAMP-dependent protein kinase activity was measured it was found to be significantly less in the inner zone than in the outer zone. The meaning of this finding, however, is unclear particularly since the activities of Ca2+-regulated protein kinases were also significantly lower in the inner zone than in the outer zone. It has been suggested that calmodulin, a protein that mediates certain intracellular actions of Ca2+, may play an important role in ACTH- stimulated steroidogenesis. Thus, the calmodulin "system" has been examined in this model. There is evidence for calmodulin kinase III activity and an endogenous substrate, elongation factor-2 (Mr 100,000). It is known that this latter system is hormonally regulated.